Semaphorin3E (sema3E) and its specific receptor plexinD1 are known to regulate the patterning of vessels during embryogenesis. However, it remains unclear whether these molecules are involved in postnatal angiogenesis. To elucidate the role of sema3E/plexinD1, we performed in vitro assay using human umbilical vein endothelial cells (HUVECs). Treatment with vascular endothelial growth factor (VEGF) increased proliferation and tube formation and this increase was significantly inhibited by sema3E. Moreover, treatment with the plexinD1-Fc fusion protein antagonized this anti-angiogenic activity of sema3E. Western blot analyses revealed that sema3E suppressed VEGF-induced phosphorylation of VEGFR2, suggesting that sema3E negatively regulates angiogenesis by inhibiting the VEGF signaling pathway. Expression of sema3E and plexinD1 was markedly upregulated in ischemic limbs. Immunohistochemistry showed that sema3E was expressed by the arterioles, myocytes, and capillary endothelial cells in ischemic tissue. Introduction of the plexinD1-Fc gene into ischemic limbs led to significant improvement of blood flow recovery and an increase in the number of CD31-positive cells. It has been reported that other members of the sema3 family are transcriptionally regulated by p53, a tumor suppressor protein that inhibits neovascularization in tumors. Consistent with these reports, forced expression of p53 was found to upregulate sema3E expression in HUVECs. We also found that the expression of p53 was markedly increased in ischemic limbs and that this increase was further enhanced in ischemic tissues of diabetic mice. Consequently, expression of sema3E was significantly higher in ischemic limbs of diabetic mice than in control mice, and the blood flow recovery after ischemia was strongly impaired in these mice even though treated with VEGF. In contrast, treatment with both VEGF and PlexinD1-Fc markedly improved blood flow recovery in diabetic mice. These results indicate that sema3E/plexinD1 negatively regulates postnatal angiogenesis under the regulation of p53 and suggest that inhibition of sema3E would be a novel strategy for therapeutic angiogenesis, especially when VEGF treatment is ineffective.